Transformation Of Amorphous Calcium Phosphate Research Articles

Role of amelogenin phosphorylation in regulating dental enamel formation

Amelogenin (AMELX), the predominant matrix protein in enamel formation, contains a singular phosphorylation site at Serine 16 (S16) that greatly enhances AMELX's capacity to stabilize amorphous calcium phosphate (ACP) and inhibit its transformation to apatitic enamel crystals. To explore the potential role of AMELX phosphorylation in vivo, we developed a knock-in (KI) mouse model in which AMELX phosphorylation is prevented by substituting S16 with Ala (A). As anticipated, AMELXS16A KI mice displayed a severe phenotype characterized by weak hypoplastic enamel, absence of enamel rods, extensive ectopic calcifications, a greater rate of ACP transformation to apatitic crystals, and progressive cell pathology in enamel-forming cells (ameloblasts). In the present investigation, our focus was on understanding the mechanisms of action of phosphorylated AMELX in amelogenesis. We have hypothesized that the absence of AMELX phosphorylation would result in a loss of controlled mineralization during the secretory stage of amelogenesis, leading to an enhanced rate of enamel mineralization that causes enamel acidification due to excessive proton release. To test these hypotheses, we employed microcomputed tomography (µCT), colorimetric pH assessment, and Fourier Transform Infrared (FTIR) microspectroscopy of apical portions of mandibular incisors from 8-week old wildtype (WT) and KI mice. As hypothesized, µCT analyses demonstrated significantly higher rates of enamel mineral densification in KI mice during the secretory stage compared to the WT. Despite a greater rate of enamel densification, maximal KI enamel thickness increased at a significantly lower rate than that of the WT during the secretory stage of amelogenesis, reaching a thickness in mid-maturation that is approximately half that of the WT. pH assessments revealed a lower pH in secretory enamel in KI compared to WT mice, as hypothesized. FTIR findings further demonstrated that KI enamel is comprised of significantly greater amounts of acid phosphate compared to the WT, consistent with our pH assessments. Furthermore, FTIR microspectroscopy indicated a significantly higher mineral-to-organic ratio in KI enamel, as supported by µCT findings. Collectively, our current findings demonstrate that phosphorylated AMELX plays crucial mechanistic roles in regulating the rate of enamel mineral formation, and in maintaining physico-chemical homeostasis and the enamel growth pattern during early stages of amelogenesis.

Composite Remineralization of Bone-Collagen Matrices by Low-Temperature Ceramics and Serum Albumin: A New Approach to the Creation of Highly Effective Osteoplastic Materials.

This study examined the effectiveness of coating demineralized bone matrix (DBM) with amorphous calcium phosphate (DBM + CaP), as well as a composite of DBM, calcium phosphate, and serum albumin (DBM + CaP + BSA). The intact structure of DBM promotes the transformation of amorphous calcium phosphate (CaP) into dicalcium phosphate dihydrate (DCPD) with a characteristic plate shape and particle size of 5-35 µm. The inclusion of BSA in the coating resulted in a better and more uniform distribution of CaP on the surface of DBM trabeculae. MG63 cells showed that both the obtained forms of CaP and its complex with BSA did not exhibit cytotoxicity up to a concentration of 10 mg/mL in vitro. Ectopic (subcutaneous) implantation in rats revealed pronounced biocompatibility, as well as strong osteoconductive, osteoinductive, and osteogenic effects for both DBM + CaP and DBM + CaP + BSA, but more pronounced effects for DBM + CaP + BSA. In addition, for the DBM + CaP + BSA samples, there was a pronounced full physiological intrafibrillar biomineralization and proangiogenic effect with the formation of bone-morrow-like niches, accompanied by pronounced processes of intramedullary hematopoiesis, indicating a powerful osteogenic effect of this composite.

Small organic molecules containing amorphous calcium phosphate: synthesis, characterization and transformation.

As the primary solid phase, amorphous calcium phosphate (ACP) is a pivotal precursor in cellular biomineralization. The intrinsic interplay between ACP and Howard factor underscores the significance of understanding their association for advancing biomimetic ACP development. While organic compounds play established roles in biomineralization, this study presents the synthesis of ACP with naturally occurring organic compounds (ascorbate, glutamate, and itaconate) ubiquitously found in mitochondria and vital for bone remodeling and healing. The developed ACP with organic compounds was meticulously characterized using XRD, FTIR, and solid-state 13C and 31P NMR. The morphological analysis revealed the characteristic spherical morphology with particle size close to 20nm of all synthesized ACP variants. Notably, the type of organic compound strongly influences true density, specific surface area, particle size, and transformation. The in vitro analysis was performed with MC3T3-E1 cells, indicating the highest cell viability with ACP_ASC (ascorbate), followed by ACP_ITA (itaconate). The lowest cell viability was observed with 10 %w/v of ACP_GLU (glutamate); however, 1 %w/v of ACP_GLU was cytocompatible. Further, the effect of small organic molecules on the transformation of ACP to low crystalline apatite (Ap) was examined in Milli-Q® water, PBS, and α-MEM.

Intrafibrillar Growth of Hydroxyapatite Nanocrystals in Multiscale Collagen

Collagen fibrils hierarchically assemble from microscale to macroscale, which endows the natural composite bone with good mechanical properties and remodeling functions. Revealing the intrafibrillar growth process of hydroxyapatite nanocrystals of collagen will guide the research of bone repair or collagen-based composites. Herein, we investigated the mineralization of multiscale collagen matrices and strongly proved the intrafibrillar hydroxyapatite nanocrystals in the collagen fibrils. The hydroxyapatite nanocrystals were deposited within collagen fibrils with co-orientation along the (002) crystal plane, which is the longitude of the fibril. The whole growth process was captured by TEM to demonstrated the five stages of the intrafibrillar growth process of hydroxyapatite nanocrystals. The infiltration and transformation of amorphous calcium phosphate in isolated collagen fibrils are both demonstrated. The intrafibrillar growth process of hydroxyapatite nanocrystals in collagen film was also investigated, showing that the growth area of collagen films increased linearly with time and the growth process. By studying the in situ mineralization under different reaction conditions, the kinetic equation of the mineralized area of collagen film under each condition was obtained, and the optimal hydroxyapatite mineralized solution was proved to be a solution with polyacrylic acid of 50 μG/mL and a pH of 7.5. Our work provides more detailed information of the growth process of HAP nanocrystals during the mineralization of collagen at different scales and would contribute to future research on the formation process of more minerals in collagen.

Comparison of bovine serum albumin and chitosan effects on calcium phosphate formation in the presence of silver nanoparticles.

The precipitation of calcium phosphates (CaPs) in the presence of more than one type of additive is of interest both from a fundamental point of view and as a possible biomimetic route for the preparation of multicomponent composites in which the activity of the components is preserved. In this study, the effect of bovine serum albumin (BSA) and chitosan (Chi) on the precipitation of CaPs in the presence of silver nanoparticles (AgNPs) stabilized with sodium bis(2-ethylhexyl)sulfosuccinate (AOT-AgNPs), poly(vinylpyrrolidone) (PVP-AgNPs), and citrate (cit-AgNPs) was investigated. In the control system, the precipitation of CaPs occurred in two steps. Amorphous calcium phosphate (ACP) was the first precipitated solid, which transformed into a mixture of calcium-deficient hydroxyapatite (CaDHA) and a smaller amount of octacalcium phosphate (OCP) after 60 min of ageing. Both biomacromolecules inhibited ACP transformation, with Chi being a stronger inhibitor due to its flexible molecular structure. As the concentration of the biomacromolecules increased, the amount of OCP decreased both in the absence and presence of AgNPs. In the presence of cit-AgNPs and two highest BSA concentrations, a change in the composition of the crystalline phase was observed. Calcium hydrogen phosphate dihydrate was formed in the mixture with CaDHA. An effect on the morphology of both the amorphous and crystalline phases was observed. The effect depended on the specific combination of biomacromolecules and differently stabilized AgNP. The results obtained suggest a simple method for fine-tuning the properties of precipitates using different classes of additives. This could be of interest for the biomimetic preparation of multifunctional composites for bone tissue engineering.

Phosphorylated and Phosphonated Low-Complexity Protein Segments for Biomimetic Mineralization and Repair of Tooth Enamel.

Biomimetic mineralization based on self‐assembly has made great progress, providing bottom‐up strategies for the construction of new organic–inorganic hybrid materials applied in the treatment of hard tissue defects. Herein, inspired by the cooperative effects of key components in biomineralization microenvironments, a new type of biocompatible peptide scaffold based on flexibly self‐assembling low‐complexity protein segments (LCPSs) containing phosphate or phosphonate groups is developed. These LCPSs can retard the transformation of amorphous calcium phosphate into hydroxyapatite (HAP), leading to merged mineralization structures. Moreover, the application of phosphonated LCPS over phosphorylated LCPS can prevent hydrolysis by phosphatases that are enriched in extracellular mineralization microenvironments. After being coated on the etched tooth enamel, these LCPSs facilitate the growth of HAP to generate new enamel layers comparable to the natural layers and mitigate the adhesion of Streptococcus mutans. In addition, they can effectively stimulate the differentiation pathways of osteoblasts. These results shed light on the potential biomedical applications of two LCPSs in hard tissue repair.

Transformation of amorphous calcium phosphate to monoclinic nano-hydroxylapatite

The transformation product of amorphous calcium phosphate without inhibitors at room temperature is monoclinic HAp rather than the more common hexagonal HAp.

Impacts of Initial Ca/P on Amorphous Calcium Phosphate

Amorphous calcium phosphate (ACP) is a metastable phase in the crystallization pathway of calcium phosphates. Understanding its chemical and structural properties could provide critical insights into biomineralization mechanisms. Of particular interest is the impact of the initial Ca/P on the structure of ACP. Further, the influence of Ca/P on the size, precipitate chemistry, and transformation of ACP is crucial to understanding the metastability of amorphous phosphates. In situ pair distribution function (PDF) analysis results 5 min after mixing show that the Ca–P bonding geometry varies from predominantly monodentate, to mixed monodentate and bidentate, to predominantly bidentate as Ca/P increases from 0.2 to 5.0. This relationship is consistent across a pH range of 6–11. Samples with only monodentate Ca–P geometries transform directly to hydroxylapatite, while samples some or all bidentate geometries form brushite. Regardless of the initial ratio, the Ca/P of precipitates is close to 1.0. In situ small-angle X-ray scattering shows particle size increases with increasing Ca/P. This is the first evidence of structural variations in ACP and is directly linked to system chemistry. Synthesis of ACP with monodentate Ca–P geometries is a promising method to control the crystallization pathway to form hydroxylapatite at circumneutral pH without stabilizing ions.

Preparation of high-performance silico-aluminophosphate geopolymers using fly ash and metakaolin as raw materials

Silico-aluminophosphate (SAP) geopolymers were synthesized by using metakaolin (MK) and a high content of fly ash (FA) for the first time. With the increase in the FA mass fraction in the raw materials from 50% to 100%, the setting time of the SAP geopolymers was shortened and their compressive strength was decreased. The effects of the FA content on the SAP geopolymers composition and microstructure were investigated. The results showed that a high FA proportion caused the lack of active aluminum, which led to excess phosphate acid reacting with the calcium from FA. The fast formation of amorphous calcium phosphate (ACP) shortened the initial setting time through the consumption of water. However, after long-term aging, ACP transformed into needle- and slice-like nanoparticles, which decreased the compressive strength of the resulting geopolymers. Moreover, MK supplied active aluminum to increase the water-containing -Si-O-Al-O-P- network (SAP gel). The SAP gel contributed to the compressive strength enhancement and prevented the ACP transformation; however, it prolonged the final setting time, due to the slow polycondensation of SAP gel. These results indicate that a desirable setting time and compressive strength of the SAP geopolymers can be achieved by adjusting the FA and MK contents.

Effect of aspartic acid on the crystallization kinetics of ACP and dentin remineralization

Type I collagen and non-collagen proteins are the main organic components of dentin. This study aimed to investigate the biomimetic remineralization of demineralized dentin by aspartic acid (Asp), which is abundant in non-collagenous proteins (NCPs). Asp was added to a mineralizing solution containing polyacrylic acid (PAA) to explore the mechanism of Asp regulating the pure amorphous calcium phosphate (ACP) phase transition process. The remineralization process and superstructure of the remineralized layer of demineralized dentin were evaluated and analyzed by transmission electron microscope (TEM) and scanning electron microscope (SEM), and the biological stability of the remineralized layer was investigated by collagenase degradation experiment. It demonstrated that Asp promoted the crystallization kinetics of PAA-stabilized amorphous calcium phosphate to hydroxyapatite (HAP), and shortened the remineralization time of demineralized dentin from 7 days to 2 days. The newly formed remineralized dentin had similar morphology and biological stability to the natural dentin layer. The presence of a large number of Asp residues in NCPs promoted the phase transformation of ACP, and further revealed the mechanism of action of NCPs in dentin biomineralization. This experiment also showed that Asp promoted the biomimetic remineralization of dentin; the morphology and hierarchical structure of remineralized layer was similar to that of natural teeth, and had good biological properties.

One-pot synthesis of amorphous calcium phosphate/Fe3O4 composites and the application in the removal of cadmium

Amorphous calcium phosphate (ACP) is a promising material for cadmium removal due to its irregular surface structure. Nevertheless, the preparation of ACP has not been trivial owing to the facile transformation of the meta-stable state of ACP into the crystalline phase of hydroxyapatite (Hap). Herein we report a new preparation, by which ACP can be synthesized at ease through a one-pot synthesis. The utility of citric acid, together with, preformed Fe3O4 renders the formation of ACP/Fe3O4 magnetic composites, hampering the aggregation of calcium phosphates and hence hindering the transformation of ACP into undesired Hap. The well-controlled syntheses are illustrated and proved to be pH-dependent. The cadmium adsorption of the ACP/Fe3O4 composite is well-fitted with Langmuir isotherm giving a maximum capacity of 454.5 mg g―1 and shows a pseudo-second order kinetic behavior. The superior cadmium adsorption capacity of the composite is top ranked among previously reported adsorbents. The thorough investigation underpins the adsorption mechanism, whereby the specific replacement of Ca2+ ions in ACP network by Cd2+ is highly selective over other M(II) ions such as Co2+ and Ni2+. Furthermore, the superparamagnetic nature of the composites allows a convenient separation of the materials after use.

Low temperature preparation of α-tricalcium phosphate up-converted luminescence nano phosphor

In this work, fluorescent α-tricalcium phosphate (α-TCP: Yb3+,Er3+) was successfully prepared by the thermal transformation of amorphous calcium phosphate (ACP) precursor. β-cyclodextrin (β-CD) was used for preparation of ACP precursor and played an important role in designing its special structure. Due to the new low temperature preparation of α-tricalcium phosphate, the obtained microsphere has a uniform distribution with rational strength (15.0 Mpa). Futhermore, the two ions (Yb3+,Er3+) were doped into α-tricalcium phosphate (α-TCP), the obtained phosphor can emit exhibit bright green luminescence under the excitation of a 980 nm laser diode. The α-TCP: Yb3+,Er3+ may have great potential in the fields of in vivo bio-imaging and a drug delivery system.

Formation of Hydroxyapatite via Transformation of Amorphous Calcium Phosphate in the Presence of Alginate Additives

Hydroxyapatite (HA) is the primary mineral of vertebral tooth and bone tissue; thus, it is often incorporated into synthetic composite materials designed for hard tissue engineering applications. U...

Bioinspired Mineralization of Type I Collagen Fibrils with Apatite in Presence of Citrate and Europium Ions

Synthetic nanostructured hybrid composites based on collagen and nanocrystalline apatites are interesting materials for the generation of scaffolds for bone tissue engineering. In this work, mineralized collagen fibrils were prepared in the presence of citrate and Eu3+. Citrate is an indispensable and essential structural/functional component of bone. Eu3+ endows the mineralized fibrils of the necessary luminescent features to be potentially employed as a diagnostic tool in biomedical applications. The assembly and mineralization of collagen were performed by the neutralization method, which consists in adding dropwise a Ca(OH)2 solution to a H3PO4 solution containing the dispersed type I collagen until neutralization. In the absence of citrate, the resultant collagen fibrils were mineralized with nanocrystalline apatites. When citrate was added in the titrant solution in a Citrate/Ca molar ratio of 2 or 1, it acted as an inhibitor of the transformation of amorphous calcium phosphate (ACP) to nanocrystalline apatite. The addition of Eu3+ and citrate in the same titrant solution lead to the formation of Eu3+–doped citrate–coated ACP/collagen fibrils. Interestingly, the relative luminescent intensity and luminescence lifetime of this latter composite were superior to those of Eu3+–doped apatite/collagen prepared in absence of citrate. The cytocompatibility tests, evaluated by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) colorimetric assay in a dose–dependent manner on GTL–16 human gastric carcinoma cells, on MG–63 human osteosarcoma cells and on the m17.ASC, a spontaneously immortalized mouse mesenchymal stem cell clone from subcutaneous adipose tissue, show that, in general, all samples are highly cytocompatible.

Transformation of amorphous calcium phosphate to bone-like apatite

Mineralisation of calcium phosphates in bone has been proposed to proceed via an initial amorphous precursor phase which transforms into nanocrystalline, carbonated hydroxyapatite. While calcium phosphates have been under intense investigation, the exact steps during the crystallisation of spherical amorphous particles to platelet-like bone apatite are unclear. Herein, we demonstrate a detailed transformation mechanism of amorphous calcium phosphate spherical particles to apatite platelet-like crystals, within the confined nanodomains of a bone-inspired nanocomposite. The transformation is initiated under the presence of humidity, where nanocrystalline areas are formed and crystallisation advances via migration of nanometre sized clusters by forming steps at the growth front. We propose that such transformation is a possible crystallisation mechanism and is characteristic of calcium phosphates from a thermodynamic perspective and might be unrelated to the environment. Our observations provide insight into a crucial but unclear stage in bone mineralisation, the origins of the nanostructured, platelet-like bone apatite crystals.

Nanogels of carboxymethyl chitosan and lysozyme encapsulated amorphous calcium phosphate to occlude dentinal tubules.

This study aimed to develop of a rapid and effective method to occlude dentinal tubules using carboxymethyl chitosan and lysozyme (CMC/LYZ) nanogels with encapsulated amorphous calcium phosphate (ACP) based on the transformation of ACP to HAP. In this work, CMC/LYZ was used to stabilize ACP and form CMC/LYZ-ACP nanogels, and then the nanogel-encapsulated ACP was applied to exposed dentinal tubule surfaces. The morphology of the nanogels was examined by transmission electron microscopy (TEM). Distribution and quantity of elements in CMC/LYZ-ACP nanogels were determined by element mapping and energy dispersive X-Ray spectroscopy (EDX). Scanning electron microscopy (SEM) images, XRD measurements and nanoindentation were applied to check the efficacy of tubular occlusion. TEM revealed that CMC/LYZ-ACP nanogels were spherical dense gel particles with size approximately 50-500 nm. Element mapping and EDX indicated that C, N, O, Ca, P, and S in the microspheres are thoroughly represented. SEM images shows that the thickness of the coating layer was approximately 1-2 μm and the depth to which the mineralized substance enters the dentinal tubule was approximately 4-8 μm. XRD measurements and nanoindentation indicated that the occluding mineralized substance observed were similar to nature dentin. CMC can form spherical dense nanogels loaded with ACP under the participation of lysozyme. The CMC/LYZ-ACP nanogels could increase the dentinal tubule occluding effectiveness. These results indicated that finding and developing novel nanomaterials of CMC/LYZ-ACP would be an effective strategy for the treatment of dentin hypersensitivity.

On Grounds of the Memory Effect in Amorphous and Crystalline Apatite: Kinetics of Crystallization and Biological Response.

Memory effects, despite being intrinsic to biological systems, are rarely potentiated in biomaterials. By exploring the transition between amorphous calcium phosphate (ACP) and hydroxyapatite (HAp) from different empirical angles, here, we attempt to set the basis for elicitation of structural memory effects in CPs. Two CPs precipitated under different degrees of saturation (DS), yielding HAp at a low DS and ACP at a high DS, were shown to evolve into structures with a high level of crystallographic similarity after their prolonged aging in the solution and served as the basis for this study. Amorphous-to-crystalline transition was abrupt in both precipitates, indicating an autocatalytic process preceded by considerable nucleation lag times, but it was more dynamic and proceeded in multiple stages in the precipitate formed at a higher DS, involving a greater degree of lattice rearrangements. ACP was found to exist in one of the two stoichiometrically and crystallographically different forms, one of which, amounting to ≥60 wt %, resembled tricalcium phosphate and transformed to HAp through the surface dissolution/reprecipitation mechanism and the other one, amounting to ≤20 wt %, was apatitic, enabling the transformation of ACP to HAp via martensitic, bulk lattice reordering phenomena. Large density of stacking faults was responsible for the comparatively high lattice strain, the property to which biogenic apatite owes its ability to accommodate foreign ions and act as a mineral reservoir for the body. Being the precursor for biogenic apatite during biomineralization and a thermodynamically logical intermediate in the ripening of HAp per the Ostwald law of stages, ACP proved to be more prone to structural transformation than the final and the most stable of the CP phases in this sequence of events: HAp. Amorphized upon gelation, two CPs transformed into HAp, albeit at different rates, which were higher for the material that had been crystalline prior to amorphization than for the one that had initially been amorphous, indicating the presence of a definite memory effect. The two HAp powders with different histories of formation also elicited different biological responses, including a Runx2 transcription factor expression in MC3T3-E1 osteoblasts, cell uptake efficiency, and antibacterial activity, extending the memory effect in HAp to the biological domain. The biological response was typically indistinct between the final products and their respective precursors but markedly different between the two products obtained by following different formation paths, confirming the presence of the given memory effect. It is suggested that the key to explaining the difference in the response between the materials differing in their route of formation lies in the direct dependence between the DS at which precipitation occurs and the rate of exchange of hydrated ions and ionic clusters across the particle surface in contact with a solution.

A Kinetic Model for Hydroxyapatite Precipitation in Mineralizing Solutions

Numerous studies have shown that there is an amorphous calcium phosphate (ACP) phase preceding the precipitation of crystalline hydroxyapatite (HA) in calcium phosphate solutions. It has also been shown that the addition of magnesium to the solutions has a stabilizing effect by inhibiting the transformation of ACP to HA. The stabilizing effect of Mg2+ is attributed to the stronger bonds between water molecules and the magnesium ions adsorbed on the surface of the ACP particles, making it harder for them to dehydrate. However, the kinetics of the reactions between calcium and phosphate ions to form ACP and then HA crystals, and the effects of varying concentrations of Mg on the kinetics have not been studied theoretically in detail. In this study, we develop and validate a kinetic model for analyzing such reactions. The pertinent rate constants are derived by calibrating the model against temporal changes in Ca2+ concentration reported by others. The predicted onset and growth of HA crystallization for sol...

The effect of serum proteins on apatite growth for 45S5 Bioglass and common sol-gel derived glass in SBF

Abstract The inhibitive effects of serum proteins on apatite growth was compared between melt-derived 45S5 Bioglass® and sol-gel derived bioactive glass of the 70S30C (70 mol% SiO2, 30 mol% CaO). By using techniques of XRD, TEM and Raman spectroscopy, the transformation of amorphous calcium phosphate to crystalline apatite, and the resulting size and aspect ratio of the crystals, in simulated body fluid (SBF), was seen to decrease in the presence of serum. XRD showed more rapid HA formation on Bioglass particles, compared to that forming on 70S30C particles, however TEM showed similar size and frequency of the needle-like crystals. Phosphate reduction in SBF was similar for Bioglass and 70S30C. Calcium carbonate formation was more likely on the phosphate-free sol-gel glass than on Bioglass.

Synthesis of β-Calcium Pyrophosphate by sol-gel method

Beta calcium pyrophosphate [β-CPP, β-Ca2P2O7] can be used as bone graft extender in posterolateral lumbar fusion. In this research, β-CPP was synthesized by sol-gel method using phosphorus pentaoxide [P2O5] and calcium nitrate tetrahydrate [Ca(NO3)2.4H2O] as phosphorus and calcium precursors. The reaction was carried out in ethanol medium with Ca/P ratio of 1.67. After 21 hours of reaction and 20 hours of drying at 80°C, white powder of amorphous calcium phosphate (ACP) was produced. Transformation of ACP to β-CPP was undertaken by firring at 400-800°C for 8 hours. Transformations of amorphous to microcrystalline, semicrystalline and crystalline structures occur at 400, 600 and 800°C, respectively. The β-CPP with the crystallite size of 61.71 nm, Ca/P ratio of 0.89 and Ca/O ratio of 0.21 was achieved by firing at 800°C. Morphology changes due to firing in which irregular shape of β-CPP at 400° changed to regular cuboid at 600 °C and above.